GPI-anchored proteins are expressed on the plasma membranes of eukaryotic cells. Their normal functions in humans range from the regulation of embryogenesis and neurogenesis to protection from complement-mediated cell destruction. Pathogenic processes involving this class of proteins include disruption of cellular architecture associated with carcinogenesis, parasitic infections, and prion-associated diseases. Understanding the trafficking of GPI-anchored proteins may lead to a further understanding of these diseases and the development of novel therapies. We explored the trafficking of native and recombinant GPI-proteins in mammalian cells. Native GPI-endocytotic pathways were examined in hematopoietic cells. Using antibodies specific for those cells and subsequent crosslinking those antibodies with fluorescently labeled avidin we demonstrated efficient endocytosis of such complexes in K562 erythroleukemia cells. We also tested the hypothesis that clustering of all surface proteins including those anchored by glycosylphosphatidylinositol can also efficiently activate endocytosis. Recombinant CD4-GPI was expressed at very high levels on human and rodent cell lines, and the direct and indirect intercellular transfer of the protein was measured by fluorescence-based assays. Membrane xenografting of the protein permitted syncytium formation with HIV gp160 expressing cells. The production of competent and attenuated HIV from those cells is currently being investigated. Based on these experiments, we plan to continue the study of GPI-protein trafficking in mammalian cells. Understanding the biology of native GPI-protein expression should enhance our understanding of recombinant GPI-protein expression in mammalian cells. The clinical applications are broad and include gene therapy and vaccine development.